右美托咪啶预处理对脐静脉内皮细胞缺氧／复氧损伤的影响及其可能机制

Effect of dexmedetomidine pretreatment on hypoxia/reoxygenation-induced injury in human umbilical vein endothelial cells and the possible mechanism

目的：探讨右美托咪啶预处理对人脐静脉内皮细胞缺氧／复氧损伤的影响及其可能机制。方法体外培养人脐静脉内皮细胞（HUVEC－12），采用随机数字表法分为4组（n＝24）：正常对照组（C组）、右美托咪定组（D组）、缺氧复氧组（H／R组）、缺氧复氧＋右美托咪定组（H／R＋D组）。D组和H／R＋D组加入50μmol／L的右美托咪定孵育2 h，随后C组和D组细胞置于常氧恒温细胞培养箱12 h，H／R组和H／R＋D组细胞缺氧6 h复氧6 h。采用四甲基偶氮唑盐微量酶反应比色（ MTT）法检测各组细胞活力，流式细胞仪检测凋亡率，逆转录聚合酶链反应（ RT－PCR）检测CHOP mRNA的表达，蛋白质印迹法（ Western blotting ）检测细胞内蛋白CHOP、cleaved caspase－3的表达量。结果与C组相比，H／R组和H／R＋D组细胞活力明显下降，细胞凋亡率、CHOP mRNA 以及CHOP蛋白和cleaved caspase－3表达升高（P＜0．05）；D组上述指标差异无统计学意义（P＞0．05）；与H／R组相比， H／R＋D组细胞活力升高，细胞凋亡率、CHOP mRNA 以及CHOP蛋白和cleaved caspase－3表达降低（P＜0．05）。结论右美托咪啶预处理可以抑制缺氧复氧引起的脐静脉内皮细胞凋亡，其机制可能与下调CHOP表达有关。

Objective To investigate the effect of dexmedetomidine pretreatment on hypoxia /reoxygenation-induced injury in human umbilical vein endothelial cells and the possible mechanism . Methods Cultured human umbilical vein endothelial cell-12（ HUVEC-12） were randomly divided into four groups（n=24）： group control （C）,group dexmedetomidine （D）,group hypoxia/reoxygenation （H/R）, group hypoxia/reoxygenation plus dexmedetomidine （ H/R ＋D ） . In groups D and H/R ＋D, Dexmedetomidine 50 μmol/L was added to the culture medium and the cells were incubated for 2 h.Then groups C and D were exposed to regular incubator and incubated for 12 h, groups H/R and H/R＋D were incubated in an anaerobic chamber for 6 h and then returned to a regular incubator and incubated for 6 h. Cell growth conditions were observed under inverted microscope , the cell viability and apoptosis were measured by methyl thiazolyl tetrazolium （ MTT） assay and flow cytometry respectively .Reverse transcriptase polymerase chain reaction （ RT-PCR） was used to detect the expression of CHOP mRNA .Western blotting was used to detect the expression of CHOP and cleaved caspase-3 protein.Results Compared with group C,the cell survival rate was significantly decreased , the expression of CHOP mRNA and CHOP protein , cleaved caspase-3 protein were up-regulated, the apoptotic rate was significantly increased in group H/R and group H/R＋D（P〈0.05）,No significant difference was found in group D （P〉0.05）;compared with group H/R,the cell survival rate was significantly increased ,the expression of CHOP mRNA and CHOP protein , cleaved caspase-3 protein were down-regulated, the apoptotic rate was decreased significantly in the group H/R＋D （ P 〈0.05 ） .Conclusions Dexmedetomidine pretreatment can effectively attenuate hypoxia /reoxygenation-induced apoptosis to human umbilical vein endothelial cell-12.The mechanism maybe related with down-regulating the expression of CHOP .